Novel listeria monocytogenes bacteriophage and uses thereof

ABSTRACT

The present invention is directed to isolated  Listeria monocytogenes  bacteriophage, and methods of using  Listeria monocytogenes  bacteriophage, or polynucleotides and polypeptides derived therefrom, to control the growth or contamination of food products by  Listeria monocytogenes . The present invention also contemplates the use of  Listeria monocytogenes  bacteriophage, and polynucleotides and polypeptides derived therefrom, for the treatment of host infections or environmental contamination by Listeria monocytogenes.

FIELD OF THE INVENTION

The present invention relates to novel bacteriophage, and compositionscorresponding thereto. More specifically, isolated Listeriamonocytogenes bacteriophage compositions are provided having lyticspecificity for Listeria monocytogenes, and are useful for controllinggrowth of Listeria monocytogenes , as well as the infection orcolonization of food products or food processing equipment by Listeriamonocytogenes, to control the infection or colonization of processed andunprocessed food products by Listeria monocytogenes, or to control thecolonization of equipment involved in the processing of the same foodproduct(s). The invention also provides methods of detecting thepresence of Listeria monocytogenes cells on processed or unprocessedfood products, or equipment involved in the processing of the same foodproducts. The invention additionally provides methods of using Listeriamonocytogenes bacteriophage for the removal of Listeria monocytogenesfrom medical, veterinary, animal husbandry, and other environments wherethey may be passed to humans or animals. The invention additionallyprovides for methods of using Listeria monocytogenes bacteriophage totreat human diseases caused by Listeria monocytogenes.

BACKGROUND OF THE INVENTION

There are six major families of bacteriophages including Myoviridae(T-even bacteriophages), Styloviridae (Lambda bacteriophage groups),Podoviridae (T-7 and related bacteriophage), Microviridae (X174 group),Leviviridae (for example, E coli bacteriophage MS2) and Inoviridae aswell as coliphages, in general. Other bacteriophage families includemembers of the Cystoviridae, Microviridae, and Siphoviridae families.

Bacteriophage has been used therapeutically for much of this century.Bacteriophage, which derive their name from the Greek word “phage”meaning “to eat” or “bacteria eaters”, were independently discovered byTwort as well as by D'Herelle in the first part of the twentiethcentury. Early enthusiasm led to the use of bacteriophage as bothprophylaxis and therapy for diseases caused by bacteria. However, theresults from early studies to evaluate bacteriophage as antimicrobialagents were variable due to the uncontrolled study design and theinability to standardize reagents. Later, in better designed andcontrolled studies, it was concluded that bacteriophage were not usefulas antimicrobial agents (Pyle, N. J., J. Bacteriol, 12:245-61 (1936);Colvin, M. G., J. Infect. Dis., 51:17-29 (1932); Boyd et al., Trans R.Soc. Trop. Med. Hyg., 37:243-62 (1944)).

This initial failure of phage as antibacterial agents may have been dueto the failure to select for phage that demonstrated high in vitro lyticactivity prior to in vivo use. For example, the phage employed may havehad little or no activity against the target pathogen, or they may havebeen used against bacteria that were resistant due to lysogenization orthe phage itself may have been lysogenic for the target bacterium(Barrow et al., Trends in Microbiology, 5:268-71 (1997)). However, withbetter understanding of the phage-bacterium interaction and of bacterialvirulence factors, it has been possible to conduct studies whichdemonstrated the in vivo anti-bacterial activity of the bacteriophage(Asheshov et al., Lancet, 1:319-20 (1937); Ward, W. E., J. Infect. Dis.,72:172-6 (1943); and Lowbury et al., J. Gen. Microbiol., 9:524-35(1953)). In the U.S. during the 1940's, Eli Lilly Co. commerciallymanufactured six phage products for human use, including preparationstargeted towards Staphylococci, Streptococci and other respiratorypathogens.

With the advent of antibiotics, the therapeutic use of phage graduallyfell out of favor in the U.S. and Western Europe, and little subsequentresearch was conducted. However, in the 1970's and 1980's bacteriophagetherapy continued to be utilized in Eastern Europe, most notably inPoland and the former Soviet Union. Alisky et al conducted a review ofall Medline citations where bacteriophage was employed therapeuticallyfrom 1966 to 1996 (Alisky et al., J. Infect., 36:5-15 (1998)).

There are also several British studies describing controlled trials ofbacteriophage raised against specific pathogens in experimentallyinfected animal models such as mice and guinea pigs (see, e.g., Smith,H. W. & M. B. Huggins, J. Gen. Microbiol. 128:307-318 (1982); Smith, H.W. & M. B. Huggins, J. Gen. Microbiol, 129:2659-2675 (1983); Smith, H.W. & R. B. Huggins, J. Gen. Microbiol., 133:1111-1126 (1987); Smith, H.W. et al., J. Gen. Microbiol., 133:1127-1135 (1987)). These trialsmeasured objective criteria such as survival rates. Efficacy againstStaphylococcus, Pseudomonas and Acinetobacter infections were observed.These studies are described in more detail below.

One such study concentrated on improving bioavailability of phage inlive animals by modifying the bacteriophage (Merril, C. R. et al., Proc.Natl. Acad. Sci. USA, 93:3188-3192 (1996)). Reports from the U.S.relating to bacteriophage administration for diagnostic purposes haveindicated phage have been safely administered to humans to monitorhumoral immune response in adenosine dearninase deficient patients (Ochset al., Blood, 80:1163-71 (1992)) and for analyzing the importance ofcell-associated molecules in modulating the immune response in humans(Ochs et al., Clin. Immunol. Immunopathol., 67:S33-40 (1993)).

Additionally, Polish, Georgian and Russian papers describe experimentswhere phage was administered systemically, topically or orally to treata wide variety of antimicrobial resistant pathogens (see, e.g.,Shabalova, I. A. et al., Abstr. 443. In Proccedings of IX InternationalCystic Fibrosis Congress, Dublin, Ireland; Slopek S. et al., Archivum.Immunol. Therapiae Experimental, 31:267-291 (1983); Slopek, S., et al.,Archivum Immunol. Therapiae Experimental, 35:569-83 (1987)).

Infections treated with bacteriophage included osteomyelitis, sepsis,empyema, gastroenteritis, suppurative wound infection, pneumonia anddermatitis. Pathogens treated with the bacteriophage includeStaphylococci, Streptococci, Klebsiella, Shigeila, Salmonella,Pseudomonas, Proteus and Escherichia. Articles have reported a range ofsuccess rates for phage therapy between 80-95% with only rare reversibleallergic or gastrointestinal side effects. These results indicate thatbacteriophage may be a useful adjunct in the fight against bacterialdiseases.

Despite the use of bacteriophage for the treatment of diseases inhumans, there remains in the art a need for the discovery of novelbacteriophage and methods for using these bacteriophage in severalcritical areas. One significant need concerns the treatment of processedor unprocessed food products to treat or prevent colonization withundesirable pathogens such as Listeria monocytogenes which isresponsible for food-borne illness. A second critical area of needconcerns the removal of undesirable bacteria from industrialenvironments such as food processing facilities to prevent colonizationthereof. A third critical area of need concerns the removal ofundesirable pathogens such as L. monocytogenes from environments wherethey may be passed to susceptible humans and animals, such assupermarkets, hospitals, nursing homes, veterinary facilities, and othersuch environments. Finally, new bacteriophage and methods of using thesame are needed for the treatment of human bacterial disease.

SUMMARY OF THE INVENTION

The invention meets those needs and more by providing compositionscomprising novel Listeria monocytogenes bacteriophage having lyticspecificity for Listeria monocytogenes. The invention additionallyprovides methods of using Listeria monocytogenes bacteriophage, tocontrol or prevent the infection or colonization of processed andunprocessed food products by Listeria monocytogenes, or colonization ofequipment involved in the processing of the same food product(s). Theinvention also provides methods of detecting the presence of Listeriamonocytogenes cells on processed or unprocessed food products, orequipment involved in the processing of the same food products. Theinvention additionally provides methods of using Listeria monocytogenesbacteriophage for the removal of antibiotic-resistant or otherundesirable pathogens from medical, veterinary, animal husbandry, andother environments where they may be passed to humans or animals. Theinvention additionally provides for methods of using Listeriamonocytogenes bacteriophage to treat human and/or other animal diseasescaused by Listeria monocytogenes.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1L show the efficacy of Listeria monocytogenes bacteriophage inreducing colonization of ready-to-eat meal and poultry products by L.monocytogenes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “isolated” will mean material removed from its originalenvironment (e.g., the natural environment in which the materialoccurs), and thus is “altered by the hand of man” from its naturalenvironment. Isolated material may be, for example, foreign nucleic acidincluded in a vector system, foreign nucleic acid contained within ahost cell, or any material which has been removed from its originalenvironment and thus altered by the hand of man. Isolated materialfurther encompasses isolated Listeria monocytogenes bacteriophage orparticular Listeria monocytogenes bacterial isolates, isolated andcultured separately from the environment in which it was located, wherethese isolates are present in purified compositions that do not containany significant amount of other bacteriophage or bacterial strains,respectively.

As used herein, “significant” will mean an amount of a substance presentin the total measured composition, wherein the substance is present ingreater than 1% of the total volume or concentration of the composition.

As used herein, “colonization” or “colonized” will refer to the presenceof Listeria monocytogenes on a foodstuff or environmental surfacewithout perceptiblc significant alteration to that foodstuff or surfaceother than the presence of bacteria. The terms “colonization” and“colonized” stand in contrast to the terms “infection” or “infected”which are commonly understood to require perceptible deleteriousalteration as part of their definition. “Colonization” and “colonized”may also refer to the presence of bacteria in or on a human or animalwithout perceptible damage, alteration, or disease.

As used herein, “ATCC” will mean the American Type Culture Collection,located at 10801 University Boulevard, Manassas, Va., 20110-2209, USA.

As used herein, “substantially pure” will mean a macromoleculeessentially free of any similar macromolecules that would normally befound with it in nature In other words, a substantially pure protein isin a composition that contains no more than 5% other proteins from thesame taxonomic species. A substantially pure composition excludes mediacomponents, excipients or other non-contaminating compounds resultingfrom culturing, processing or formulating the composition.

As used herein, “amplification” will mean the in vitro production ofmultiple copies of a particular nucleic acid sequence. The amplifiedsequence is usually in the form of DNA. A variety of techniques forcarrying out such amplification are described in a review article by VanBrunt (1990, Bio/Technol., 8(4):291-294). Polymerase chain reaction orPCR is a prototype of nucleic acid amplification, and use of PCR hereinshould be considered exemplary of other suitable amplificationtechniques. Other forms of amplification include, but are not limitedto, ligase chain reaction (LCR) and gap-LCR.

Listeria monocytogenes Bacteriophage

The invention provides novel Listeria monocytogenes bacteriophageparticles. In particular, this invention provides isolated Listeriamonocytogenes bacteriophage List-1, List-2, List-3, List-4, List-36 andList-38, deposited with the ATCC and receiving ATCC Deposit AccessionNos. PTA-5372, PTA-5373, PTA-5374, PTA-5375, PTA-5376 and PTA-5377,respectively. Unless otherwise indicated, use of the term “Listeriamonocytogenes bacteriophage” in this application is intended toencompass each of the deposited bacteriophage, or mixtures of one ormore, up to all of them.

Listeria monocytogenes bacteriophage has binding specificity forListeria monocytogenes, and is capable of lysing many infected hostListeria monocytogenes cells. Particularly preferred Listeriamonocytogenes bacteriophage have biological activity (e.g., the abilityto lyse host Listeria monocytogenes cells and/or the ability to producephage progeny in a host cell). The invention further contemplates“variants” of Listeria monocytogenes bacteriophage, which arebacteriophage having minor variation(s) in the genomic sequence andpolypeptides encoded thereby while retaining the same general genotypicand phenotypic characteristics as the Listeria monocytogenesbacteriophage. Variants of Listeria monocytogenes bacteriophageencompass polymorphic variants. The invention also contemplates“derivative” bacteriophage, which are bacteriophage having modifiedgenotypic or phenotypic characteristics relative to the depositedListeria monocytogenes bacteriophage. Derivative bacteriophage of theinvention particularly encompass recombinantly designed Listeriamonocytogenes bacteriophage harboring genes encoding novel phenotypictraits. Such recombinant Listeria monocytogenes bacteriophage areengineered to contain novel genes having traits not found in wild-typeListeria monocytogenes bacteriophage. Variant Listeria monocytogenesbacteriophage capable of performing the same or equivalent biologicalfunctions as Listeria monocytogenes bacteriophage are particularlypreferred.

The invention contemplates the use of Listeria monocytogenesbacteriophage, or variants thereof to control the growth on, orcolonization of, processed and unprocessed food products by Listeriamonocytogenes , or the colonization of buildings and equipment,particularly those associated with the processing of the same foodproduct. The invention also provides methods of detecting the presenceof Listeria monocytogenes cells on processed or unprocessed foodproducts, or equipment or buildings such as those involved in theprocessing of the same food products. The invention further providesmethods of using Listeria monocytogenes bacteriophage for the removal ofantibiotic-resistant or other undesirable pathogens from medical,veterinary, animal husbandry, or any additional environments where theymay be passed to humans or animals. The invention additionally providesfor methods of using Listeria monocytogenes bacteriophage to treat humanand animal diseases caused by Listeria monocytogenes. Listeriamonocytogenes bacteriophage are administered for the methods of theinvention as a homogenous phage administration, or alternatively as acomponent of a multi-phage composition comprising numerous, relatedbacteriophage, all having lytic specificity for at least one Listeriamonocytogenes strain. These methods of use are provided with greaterparticularity infra.

Use of Listeria monocytogenes Bacteriophage

Food Preservation

In one embodiment, the invention contemplates a method for theprevention of food borne illnesses caused by the bacterium Listeriamonocytogenes, comprising contacting a food product or products with amicrobial growth inhibiting effective amount of a bacteriophagecomposition comprising Listeria monocytogenes bacteriophage. The modesof contact include, but are not limited to, spraying or misting theListeria monocytogenes bacteriophage composition on the food product(s),or by dipping or soaking the food product(s) in a solution containing aconcentration of Listeria monocytogenes bacteriophage sufficiently highto inhibit the growth of Listeria monocytogenes or adding, injecting orinserting Listeria monocytogenes bacteriophage into the food product(s).

In another embodiment, the invention contemplates the application of aListeria monocytogenes bacteriophage composition to equipment associatedwith the processing of food product(s), such as cutting instruments,conveyor belts, and any other implements utilized in the mass productionof food products, including the preparation, storage lad packaging stepsof food processing. Listeria monocytogenes bacteriophage canadditionally be introduced into packaging materials used to contain foodproduct(s), prior to or following transfer of the food product(s) to thepackaging materials. Alternatively Listeria monocytogenes bacteriophageis useful in the local processing of food products (e.g., in the home orin the restaurant kitchen), using the same modes of contact as describedsupra.

In another embodiment of the invention, Listeria monocytogenesbacteriophage are added as a component of paper products, either duringprocessing or after completion of processing of the paper products.Paper products to which Listeria monocytogenes bacteriophage may beadded include, but are not limited to, paper towels, toilet paper, moistpaper wipes. In a preferred embodiment of the invention, Listeriamonocytogenes bacteriophage are added as a component of cleansing wipes.Listeria monocytogenes bacteriophage may be added in an aqueous state toa liquid-saturated paper product, or alternatively may be added inpowder furixi (e.g., lyophilized) to dry paper products, or anycombination thereof In similar manner, Listeria monocytogenesbacteriophage may be incorporated into films such as those used forpackaging foods, e.g., by impregnating or coating the film.

The methods of the invention further contemplate the application ofListeria monocytogenes bacteriophage to the floors, walls, ceilings,drains, or other environmental surfaces in structures such as theindustrial food processing or home environments. In a particularlypreferred embodiment of the invention, Listeria monocytogenesbacteriophage is applied to refrigerated devices used to store ortransport food or food products, including but not limited to, home andindustrial refrigerators, deli meat and cheese counters, refrigeratedtrucks, and mobile food service vehicles.

In a non-limiting embodiment of the invention, Listeria monocytogenesbacteriophage of the invention are useful in preventing the colonizationof, or inhibiting the growth of, Listeria monocytogenes on processed orunprocessed food products by infecting, lysing or inactivating Listeriamonocytogenes present on said food product.

Processed or unprocessed food products in which Listeria monocytogenesbacteriophage are particularly useful in preventing the growth orcolonization of Listeria monocytogenes include, but are not limited to,hot dogs, deli meals, luncheon meals, soft cheeses such as feta, brie,camembert, blue-veined cheeses, Mexican-style cheeses, pates, meatspreads, smoked seafoods such as salmon, trout, whitefish, cod, tuna ormackerel, poultry, salads, eggs, milk and dairy products, fish, shrimp,frog legs, yeast, coconut, sauces and salad dressing, cake mixes,cream-filled desserts and toppings, dried gelatin, peanut butter,chocolate and ground beef.

Listeria monocytogenes bacteriophage can also be administered withready-to-eat foods and food products such as frankfurters and sliceddeli meats including both meat and poultry products as well as wholemuscle, sliced, and comminuted products,

Additional “ready to eat” foods to which Listeria monocytogenesbacteriophage may be administered include, but are not limited to,cooked cured comminuted red meat products (such as beef and porkfrankfurters); cooked cured comminuted poultry products (such as turkeyfrankfurters and chicken bologna); sliced cooked whole red meat musclecuts, uninjected (such as sliced roast beef and sliced fresh hamprepared from minimally processed cuts); sliced cooked whole poultrymuscle cuts, uninjected (such as sliced turkey breast and sliced chickenbreast prepared from minimally processed cuts); sliced cooked curedwhole red meat muscle cuts (such as corned beef and pastrami); slicedcooked cured whole poultry muscle cuts (such as turkey pastrami);injected whole red meat muscle cuts (such as barn and most processedand/or flavored whole muscle roast beef products); and injected wholepoultry muscle cuts (such as most processed and/or flavored whole musclechicken and turkey breast products.

Listeria monocytogenes bacteriophage can also be administered to potableand non-potable water sources to reduce or eliminate the presence ofListeria monocytogenes.

Listeria monocytogenes bacteriophage compositions of the invention maybe provided in aqueous or non-aqueous embodiments for the preservationof food. Aqueous embodiments of Listeria monocytogenes bacteriophageinclude aqueous compositions comprising, or alternatively consisting of,Listeria monocytogenes bacteriophage alone or in combination with otherbacteriophage. Other bacteriophage include either bacteriophage specificfor Listeria monocytogenes or bacteriophage specific for other bacterialspecies, or both. Aqueous embodiments of Listeria monocytogenesbacteriophage are available in solutions that include, but are notlimited to, phosphate buffered saline, Luria-Bertani broth orchlorine-free water.

Non-aqueous embodiments of Listeria monocytogenes bacteriophage include,but are not limited to, lyophilized compositions or spray-driedcompositions comprising, or alternatively consisting of, Listeriamonocytogenes bacteriophage alone or in combination with otherbacteriophage.

Listeria monocytogenes bacteriophage can be administered at aconcentration effective to inhibit the growth or colonization of food orfood products, as well as the equipment used to process or store food.In a non-limiting embodiment of the invention, Listeria monocytogenesbacteriophage are typically administered at a growth inhibitingeffective amount of a concentration of about 10⁷ to about 10¹¹ PlaqueForming Units (PFU)/ml, and most preferably at 10⁹ PFU/ml. One of skillin the art is capable of ascertaining bacteriophage concentrations usingwidely known bacteriophage assay techniques. Listeria monocytogenesbacteriophage at such concentrations may be applied at, for example, 1ml per 500 cm² of food product.

Environmental Control

In another embodiment of the invention, Listeria monocytogenesbacteriophage compositions are administered to environments to controlthe growth or viability of Listeria monocytogenes, particularly thegrowth or viability of antimicrobial resistant strains of Listeriamonocytogenes. Antimicrobial resistant Listeria monocytogenes include,but are not limited to, Listeria monocytogenes showing resistance toampicillin, amoxicilliniclavulanic acid, chloramphenicol,sulfamethoxazole/trimethoprim, ciprofloxacin, fluoroquinolones,enrofloxacin, clindamycin, penicillin, tetracycline pediocin PA-1, nisinA and cephalosporins. Environments in which Listeria monocytogenesbacteriophage is useful to control the growth or viability of Listeriamonocytogenes include, but are not limited to, medical facilities(including hospitals, out-patient clinics, school and/or universityinfirmaries, and doctors offices), veterinary offices, animal husbandryfacilities, public and private restrooms, and nursing and nursing homefacilities. The invention further contemplates the use of Listeriamonocytogenes bacteriophage for the battlefield decontamination of foodstuffs, the environment, and personnel and equipment, both military andnon-military.

Listeria monocytogenes bacteriophage are additionally useful alone or incombination with other bacteriophage or other compounds, for controllingthe growth of biofilms in aquatic environments. Other bacteriophageinclude either bacteriophage specific for Listeria monocytogenes ofbacteriophage specific for other bacterial species, or both. Aqueousembodiments of Listeria monocytogenes bacteriophage are available insolutions that include, but are not limited to, phosphate bufferedsaline, Luria-Bertani broth or chlorine-free water. In a particularlypreferred embodiment, Listeria monocytogenes bacteriophage is used tocontrol biofilm growth in municipal and personal water systems, as wellas biofilms present in refrigerated environments.

The modes of administration include, but are not limited to, spraying,hosing, and any other reasonable means of dispersing aqueous ornon-aqueous Listeria monocytogenes bacteriophage compositions, in anamount sufficiently high to inhibit the growth or viability of Listeriamonocytogenes. In a non-limiting embodiment of the invention, Listeriamonocytogenes bacteriophage are useful in preventing the growth orviability of Listeria monocytogenes by infecting, lysing or inactivatingListeria monocytogenes present in said environment. Administration ofthe Listeria monocytogenes bacteriophage composition includesapplication to the floors, walls, counter-tops, ceilings, drains or anyother environmental surface.

Listeria monocytogenes bacteriophage compositions of the invention areavailable in aqueous or non-aqueous embodiments for the treatment ofvarious environments. Aqueous embodiments of Listeria monocytogenesbacteriophage include aqueous compositions comprising, or alternativelyconsisting of, Listeria monocytogenes bacteriophage alone or incombination with other bacteriophage. Aqueous embodiments of Listeriamonocytogenes bacteriophage are available in solutions that include, butare not limited to, phosphate buffered saline or chlorine-free water.

Non-aqueous embodiments of Listeria monocytogenes bacteriophage include,but are not limited to, lyophilized compositions or spray-driedcompositions comprising, or alternatively consisting of, Listeriamonocytogenes bacteriophage alone or in combination with otherbacteriophage. Spray-dried compositions may include soluble and/orinsoluble carrier materials as processing aids.

In another embodiment of the invention, Listeria monocytogenesbacteriophage are added as a component of paper products, either duringprocessing or after completion of processing of the paper products.Paper products to which Listeria monocytogenes bacteriophage may beadded include, but are not limited to, paper towels, toilet paper andmoist paper wipes. In a preferred embodiment of the invention, Listeriamonocytogenes bacteriophage are added as a component of cleansing wipes.Listeria monocytogenes bacteriophage may be added in an aqueous state toa liquid-saturated paper product, or alternatively may be added inpowder form (e.g., lyophilized) to dry paper products, or anycombination thereof.

Listeria monocytogenes bacteriophage can be administered at aconcentration effective to inhibit the growth or viability of Listeriamonocytogenes in a particular environment. In a non-limiting embodimentof the invention, Listeria monocytogenes bacteriophage are administeredat a concentration of about 10⁷ to 10¹¹ PFU/ml. One of skill in the artis capable of ascertaining bacteriophage concentrations using widelyknown bacteriophage assay techniques.

Prevention or Treatment of Infection

In another embodiment, the invention contemplates a method for theprevention or treatment of illnesses caused by the bacterium Listeriamonocytogenes, comprising contacting a microbial growth inhibitingeffective amount of a bacteriophage composition comprising Listeriamonocytogenes bacteriophage with a site or sites of infection of a hostmammal infected with Listeria monocytogenes.

The infected mammalian host may be a human host. Listeria monocytogenestreatment of infected persons is particularly preferred in the treatmentof immuno-compromised persons, pregnant females, and newborns andinfants, who are all at an elevated risk of infection by Listeriamonocytogenes. The modes of contact include, but are not limited to,spraying or misting the Listeria monocytogenes bacteriophage compositionon the infected mammalian host, by injecting at a site or sites ofinfection a pharmaceutically acceptable composition containing aconcentration of Listeria monocytogenes bacteriophage sufficiently highto inhibit the growth of Listeria monocytogenes, or by ingesting asolution containing a concentration of Listeria monocytogenesbacteriophage sufficiently high to inhibit the growth of Listeriamonocytogenes. Additional routes of administration include but are notlimited to oral, rectal, topical, ophthalmic, buccal, intravenous,optic, nasal, vaginal, inhalation and intrapleural. The composition isformulated as known in the pharmaceutic arts.

Listeria monocytogenes bacteriophage compositions of the invention areavailable in aqueous or non-aqueous embodiments for the treatment ofinfection. Aqueous embodiments of Listeria monocytogenes bacteriophageinclude aqueous compositions comprising, or alternatively consisting of,Listeria monocytogenes bacteriophage alone or in combination with otherbacteriophage. Aqueous embodiments of Listeria monocytogenesbacteriophage are available in solutions that include, but are notlimited to, phosphate buffered saline or chlorine-free water.

Non-aqueous embodiments of Listeria monocytogenes bacteriophage include,but are not limited to, lyophilized compositions or spray-driedcompositions comprising, or alternatively consisting of, Listeriamonocytogenes bacteriophage alone or in combination with otherbacteriophage. Spray-dried compositions may include soluble and/orinsoluble carrier materials as processing aids.

Listeria monocytogenes bacteriophage can be administered at aconcentration effective to inhibit the growth or viability of Listeriamonocytogenes in the infected host. In a non-limiting embodiment of theinvention, Listeria monocytogenes bacteriophage are administered at aconcentration of about 10⁷ to 10¹¹ PFU/ml. One of skill in the art iscapable of ascertaining bacterlophage concentrations using widely knownbacteriophage assay techniques.

Depending on the severity of peculiarities of the infection, Listeriamonocytogenes bacteriophage can be administered to humans (i) orally, intablet or liquid formulation (10⁵-10¹¹ PFU/dose), (ii) rectally, (iii)locally (skin, eye, ear, nasal mucosa, etc.), in tampons, rinses andcreams, (iv) as aerosols or intrapleural injections, (v) intravenouslyand (vi) intrathecally. Most preferably, bacteriophage can beadministered for treatment of L monocytogenes infections by oral orrectal routes.

Production of Listeria monocytogenes Bacteriophage

Listeria monocytogenes bacteriophage are produced using a culturesystem. More specifically, host Listeria monocytogenes are cultured inbatch culture, followed by inoculation of the Listeria monocytogenesculture with an appropriate inoculum of Listeria monocytogenesbacteriophage. Following incubation, the Listeria monocytogenesbacteriophage are harvested and filtered to yield phage progeny suitablefor the uses enumerated herein.

The invention provides compositions comprising active viral particles ofListeria monocytogenes bacteriophage capable of lysing Listeriamonocytogenes strains.

The concentration of Listeria monocytogenes bacteriophage may bedetermined using phage titration protocols. The final concentration ofListeria monocytogenes bacteriophage can be adjusted by dilution withbuffer to yield a phage titer of 10⁹ to 10¹¹ PFU/ml. The resultingListeria monocytogenes bacteriophage composition can be freeze-dried orspray-dried for storage. On reconstitution, the phage titer can beverified using phage titration protocols and host Listeria monocytogenesbacteria. One of skill in the art is capable of determiningbacteriophage titers using widely known bacteriophage assay techniques(e.g., Davis et al., “Microbiology” 3^(rd) ed., Harper & Row,Hagerstown, 1980, pp. 874-877, 880-883).

Polynucleotides

Polynucleotides and Variants thereof

The invention contemplates isolated polynucleotide molecules of theListeria monocytogenes bacteriophage, contained within bacteriophagedeposits submitted with the ATCC and receiving ATCC Deposit AccessionNos. PTA-5372, PTA-5373, PTA-5374, PTA-5375, PTA-5376 and PTA-5377.

Polynucleotides of the invention encompass polyribonucleotide andpolydeoxyribonucleotide, including modified or unmodified RNA or DNA.Polynucleotides of the invention can derive from genomic DNA, as well ascDNA, mRNA and synthetic polynucleotide sequences. One of ordinary skillin the art is well aware of techniques for generating cDNA sequence frommRNA sequence. Polynucleotides of the invention comprise single ordouble-stranded DNA or RNA sequences, as well as DNA/RNA hybrids.

Polynucleotides of the invention also encompass modifiedpolynucleotides, such as for example phosphorothioated DNAs or PNAs(Peptide Nucleic Acids). Additionally, polynucleotides of the inventionmay include one or more labels (e.g., radioactive label, biotin,fluorescent label, chemiluminescent or colorimetric label) fordiagnostic or tracking and monitoring purposes.

Polynucleotide Fragments

The invention further contemplates fragments of the polynucleotidesdiscussed supra. Polynucleotide fragments are particularly useful forthe detection of Listeria monocytogenes bacteriophage. Using DNAisolation techniques known in the art or described herein (i.e., CsClgradients and pulsed field gel electrophoresis), one of skill is capableof using the polynucleotide isolation techniques to obtain Listeriamonocytogenes bacteriophage DNA, from which polynucleotide fragments aregenerated. Numerous techniques for generating polynucleotide fragmentsare also widely known in the art (e.g., restriction digests,pressure-shearing via French Press etc.). Fragments can be isolated viagel electrophoresis or other means and radioactively ornon-radioactively labeled for use as probes. DNA fragments can also bepurified using HPLC. Labeled polynucleotide fragments are useful understringent hybridization conditions to identify Listeria monocytogenesbacteriophage from a bacteriophage culture or environmental surface.Kits are widely available in the art for labeling polynucleotidefragments (see Invitrogen product catalog and Sigma-Aldrich productcatalog).

Polypeptides

Polypeptides and Variants thereof

The invention further encompasses polypeptides encoded by thepolynucleotides of the invention, contained within ATCC DepositAccession Nos. PTA-5372, PTA-5373, PTA-5374, PTA-5375, PTA-5376 andPTA-5377. Polypeptides of the invention may encompass viral coatproteins, transcriptional regulatory proteins and virulence proteins.

Polypeptides of this invention are molecules having an amino acidsequence encoded by polynucleotides of the invention as broadly defined.Polypeptides encompasses proteins, peptides and fragments thereof(functional or non-functional) encoded by Listeria monocytogenesbacteriophage polynucleotides. Preferred polypeptides of the inventioncomprise, or alternatively consist of, antigenic and/or immunogenicpolypeptides, especially antigenic and/or immunogenic polypeptidefragments.

Derivative Listeria monocytogenes Bacteriophage

Polynucleotides of the invention are also useful for the production ofderivative Listeria monocytogenes bacteriophage, particularlyrecombinant Listeria monocytogenes bacteriophage. In one embodiment ofthe invention, homologous recombination techniques are used to introducehomologous sequences encoding alternative proteins, non-functionalproteins, or non-coding sequences into the Listeria monocytogenesbacteriophage DNA sequence. Such techniques are useful to “knock-out”undesired traits of the Listeria monocytogenes bacteriophage, oralternatively to introduce different traits. In a particularly preferredembodiment of the invention, homologous recombination is used to“knock-out” ORFs encoding proteins that are putatively involved in alysogenic cycle of the Listeria monocytogenes bacteriophage.

In another embodiment of the invention, the invention providesrecombinant Listeria monocytogenes bacteriophage having novelbacteriophage genes introduced into the Listeria monocytogenesbacteriophage sequence. In this embodiment, the double-crossover(homologous recombination) method of Loessner et al. (incorporatedherein by reference in its entirety) is utilized to introduce a novelbacteriophage gene(s) into the genome of Listeria monocytogenesbacteriophage. Successful recombinant Listeria monocytogenesbacteriophage replicate in the host Listeria monocytogenes cell,producing recumbinant progeny phage.

In certain embodiments of the invention it is important to confirm thatbacteriophage cocktails contain “lytic” phage rather than “lysogenic”phage, as some lysogenic phage (i.e., transducing phage) may be capableof transferring “undesirable” bacterial genes (e.g., genes encodingbacterial toxins) from one bacterial host to another. Therefore, the useof lysogenic phage on an industrial scale could increase the risk ofacquisition of “undesirable” genes from new bacterial strains, whichcould contribute to the emergence of new pathogenic bacteria. It istherefore prudent to make efforts to avoid or to minimize the use ofphage, either in agribusiness or in human therapeutic settings, that (i)contain genes directly associated with bacterial virulence (so thatadditional virulence genes are not introduced into the environment)and/or (ii) can significantly contribute to the horizontal transfer ofvirulence-associated genes between bacterial species or strains (tominimize the risk of phage-mediated transduction of undesirable genes).Accordingly, in an alternative embodiment of the invention, homologousrecombination is used to “knock-out” undesirable genes such as bacterialtoxin genes, or genes having significant homology thereto, found inListeria monocytogenes bacteriophage DNA. A list of undesirablebacterial toxin genes is provided in Table 1. Additional undesirablebacterial genes are listed in 40 CFR §725.421, which is incorporatedherein by reference.

In another embodiment of the invention, homologous recombination is usedto introduce or knock-out genes involved in burst size. For example,homologous recombination is used to introduce alternative bacteriophagegenes which delay the burst event or increase the phage burst size.

References disclosing alternative bacteriophage genes involved in thetiming of the burst event or the size of the phage burst include, butare not limited to, Wang I. N. et al. (2000), Annu. Rev. Microbiol.;54:799-825; and Johnson-Boaz R. et al. (1994), Mol. Microbiol.,13(3):495-504.

Recombinant Listeria monocytogenes Bacteriophage Reporter Systems

In another embodiment of the invention, recombinant Listeriamonocytogenes bacteriophage harboring a reporter system(s) are generatedusing polynucleotides of the invention. L. monocytogenes bacteriophagereporter systems of the invention are useful for the detection of thepresence of viable L. monocytogenes cells to which the bacteriophagehave specificity. Following the technique of Loessner et al., forexample, one of skill in the art can generate recombinant L.monocytogenes reporter bacteriophage (Loessner et al., Appl. Environ.Micro., 62(4):1133-1140 (1996)). For example, the Vibrio harveyi luxABgene may be introduced into the L. monocytogenes bacteriophage DNAsequence using techniques such as homologous recombination. An idealtarget for the introduction of the luxAB gene is immediately downstreamand in frame with an ORF encoding a L. monocytogenes bacteriophagecapsid protein, thereby creating a sequence encoding a fusion protein.The preferable location of introduction of the luxAB gene sequence isparticularly before any sequence encoding a transcriptional terminatordownstream of the ORF encoding a capsid protein. Other L. monocytogenesbacteriophage ORF sequences which may function as useful sources ofluxAB gene-fusions include gene sequences encoding tail-sheath proteins,or any other late gene region sequences encoding phage head or tailproteins. Such information can be determined using the polynucleotidesisolated from ATCC Deposit Accession Nos. PTA-5281, PTA-5284, PTA-5282,PTA-5285, PTA-5283 and PTA-5280 and obtaining and analyzing sequencedata derived therefrom. Recombinant polynucleotides harboring thereporter gene are used to generate progeny phage harboring the reportergene, and expressing the reporter gene-fusion.

TABLE 1 Undesirable (e.g., Bacterial toxin) Genes known to be carried byTransducing Bacteriophages Toxin and its Encoding Gene BacterialPathogen Reference Enterotoxin A (entA) Staphylococcus aureus Betley andMekalanos, 1988 Enterotoxin A (sea, sel) Staphylococcus aureus Betleyand Mekalanos, 1985 Enterotoxin A (sea) Staphylococcus aureus Kuroda etal., 2001 Staphylokinase (sak) Staphylococcus aureus Coleman et al.,1989 Enterotoxin P (sep) Staphylococcus aureus Kuroda et al., 2001Exfoliative toxin A (eta) Staphylococcus aureus Yamaguchi et al., 2000Diphtheria toxin (tox) Corynebacterium diphtheriae Freeman, 1951 Shigatoxins (stx1,2) Escherichia coli O'Brien et al., 1984 Cytotoxin (ctx)Pseudomonas aeruginosa Nakayama et al., 1999 Cholera toxin (ctxA) Vibriocholerae Waldor & Mekalanos, 1996 Cholera toxin (ctxB) Vibrio choleraeWaldor & Mekalanos, 1996 Zonula occludens toxin (zot) Vibrio choleraeKoonin, 1992 Neurotoxin (C1) Clostridium botulinum Barksdale and Arden,1974. Enterohaemolysin (hly) Escherichia coli Beutin et al., 1993Streptococcal exotoxin A (speA) Streptococcus pyogenes Weeks andFerretti, 1984 Streptococcal exotoxin C (speC) Streptococcus pyogenesGoshorn and Schlievert, 1989 Streptococcal exotoxin K (speK)Streptococcus pyogenes Beres et al., 2002

Successful recombinant phage are subsequently screened using aluciferase assay in which L. monocytogenes bacteriophage (in lysates,for example) containing the luciferase-reporter fusion protein are mixedwith a L. monocytogenes culture, and cultured for a fixed period of time(e.g., 90 to 120 minutes). Samples are then assayed for bioluminscenceusing a tube luminometer. Successful recombinant L. monocytogenesbacteriophage expressing the reporter fusion protein in the presence ofviable L. monocytogenes are isolated and cultured to appropriateconcentrations to allow for the isolation and storage of saidrecombinant bacteriophage. The resulting recombinant L. monocytogenesbacteriophage may be used with methods of the invention to detect thepresence of viable L. monocytogenes.

In addition to the Vibrio harveyi luxAB gene, other reporter genes whichare useful for the generation of L. monocytogenes reporter bacteriophageinclude, but are not limited to, the firefly luciferase gene.

The invention further contemplates the introduction of one or more ofthe above-described recombinant events. For example, a recombinant L.monocytogenes bacteriophage of the invention may harbor a reporter geneas well as lack a gene associated with the lysogenic cycle.

Use of Listeria monocytogenes Bacteriophage Polynucleotides andPolypeptides Therefrom

Polypeptides such as Listeria monocytogenes bacteriophage lytic enzymesencoded by polynucleotides of the invention are used for applicationsdesigned to prevent the growth of Listeria monocytogenes through cellwall lysis. Thus, lytic polypeptides are useful for the prevention ofthe growth of Listeria monocytogenes on processed and unprocessed foodproducts, as well as equipment used for the processing of said foodproducts.

In another preferred embodiment of the invention, Listeria monocytogenesbacteriophage lytic polypeptides are useful for the treatment of one ormore infections in a mammal, including humans, by administering atherapeutically effective amount of a Listeria monocytogenesbacteriophage lytic enzyme to the patient. This method is useful for thetreatment of Listeria monocytogenes infections of the gastrointestinalsystem. Similarly, this method is useful in a prophylactic setting forthe prevention of infection by Listeria monocytogenes in infant,pregnant or aged mammals, including humans. This method of treatment isfurther useful for the prevention or other disorders or infectionscaused by Listeria monocytogenes, including but not limited to Listeriameningitis and brain abscesses caused by Listeria monocytogenes.

Detection Systems

Listeria monocytogenes bacteriophage polynucleotides are particularlypreferred in a method of detecting the presence of Listeriamonocytogenes bacteriophage. For example, fragments of at least 20nucleotides in length are useful as probes for the identification of thepresence of Listeria monocytogenes bacteriophage in an environmental orfood sample using hybridization techniques. Using stringenthybridization techniques as known in the art, one skilled in the art candetermine the presence of Listeria monocytogenes bacteriophage in asample.

In another embodiment of the invention, polynucleotide fragments ofbetween about 16 and about 40 nucleotides in length are useful asprimers for the identification of the presence of Listeria monocytogenesbacteriophage in, e.g., an environmental or food sample using PCRamplification techniques. These applications are particularly useful inthe sense of determining the presence of Listeria monocytogenesbacteriophage in food over extended periods of time following treatmentof the food with Listeria monocytogenes bacteriophage. PCR amplificationconditions may vary, but one skilled in the art can readily determinethe appropriate PCR amplification conditions (see, e.g., CurrentProtocols in Molecular Biology, Frederick M. Ausubel et al., ed.,Wiley-Interscience, NY, 1989 and periodic updates thereof).

Alternatively, recombinant Listeria monocytogenes bacteriophagethemselves, such as, for example, the Listeria monocytogenes luciferasereporter bacteriophage described supra, are useful in methods ofscreening food products and food processing equipment for the presenceof viable Listeria monocytogenes. In such a system, Listeriamonocytogenes bacteriophage containing a reporter system (such as, forexample, a luciferase reporter system) are applied to the sample andanalyzed at some time point in the future for the activation of thereporter molecule. The activation of the reporter molecule is indicativeof the presence of viable Listeria monocytogenes cells.

In a preferred embodiment of the invention, Listeria monocytogenesbacteriophage polynucleotides or fragments thereof are useful as probesto detect the presence of Listeria monocytogenes bacteriophage. Inanother embodiment of the invention, Listeria monocytogenesbacteriophage polynucleotides or fragments thereof are useful as part ofa process for the detection of Listeria monocytogenes bacteriophageduring production of the same. Alternatively, Listeria monocytogenesbacteriophage polynucleotides or fragments thereof are useful for thedetection of the presence of Listeria monocytogenes bacteriophageintroduced into foodstuffs or packaging materials for the same duringpart of a production method for the production or packaging of foodstuffs. In an additional embodiment of the invention, more than onelabeled Listeria monocytogenes bacteriophage polynucleotide fragment isused as a probe to detect the presence of Listeria monocytogenesbacteriophage in a sample. Polynucleotide fragments of the inventionuseful for the detection of Listeria monocytogenes bacteriophage arepreferably at least 20 nucleotides in length. Polynucleotide fragmentsof the invention are also useful for the detection of closely relatedListeria monocytogenes bacteriophage isolates under stringent ornon-stringent hybridization conditions. Polynucleotide fragments of theinvention may include one or more labels (e.g., radioactive label,biotin, fluorescent label, chemiluminescent or calorimetric label) fordiagnostic or tracking and monitoring purposes.

In another embodiment of the invention, polynucleotides and polypeptidesof the invention, or fragments thereof, are used in techniques toidentify Listeria monocytogenes bacteriophage. By way of the followingnon-limiting list of experimental techniques, one skilled in the art caneasily identify bacteriophage compositions as comprising Listeriamonocytogenes bacteriophage when the same techniques are performed on acomparative basis against the bacteriophage deposited in ATCC DepositAccession Nos. PTA-5372, PTA-5373, PTA-5374, PTA-5375, PTA-5376 andPTA-5377. The experimental techniques that can be used include, but arenot limited to, DNA sequencing; polymerase chain reaction (PCR) withsequence-specific primers; Southern blot DNA hybridization withsequence-specific nucleic acid probes; restriction fragment lengthpolymorphism (RFLP) analysis; SDS polyacrylamide gel electrophoresisanalysis of raw protein extracts; SDS polyacrylamide gel electrophoresisanalysis of raw protein extracts with protein sequencing by any meansavailable; peptide mapping experiments; 2D-gel electrophoresis profiles,and Western blot analysis. These and other useful techniques are fullyenabled by the deposited bacteriophage in view of the presentspecification and laboratory references such as “Current Protocols inMolecular Biology,” Frederick M. Ausubel et al., ed.,Wiley-lnterscience, N.Y., 1989 and periodic updates thereof; Sambrook etal., “Molecular Cloning: A Laboratory Manual,” 3rd ed., 2001; andColigan et al., eds., “Current Protocols in Protein Science,” Wiley,Brooklyn, N.Y., 2001 and periodic updates thereof, each of which areincorporated herein by reference.

Epidemiological Typing

Listeria monocytogenes bacteriophage of the invention are further usefulas a tool for the epidemiological typing of Listeria monocytogenesisolates. For example, one of skill in the art can use Listeriamonocytogenes bacteriophage of the invention to screen a panel ofListeria monocytogenes isolates to aid in the taxonomic identificationof the Listeria monocytogenes, by determining which isolates yield apositive lytic reaction to the Listeria monocytogenes bacteriophage.(see, for example, Mee-Marquet et al. Appl. Env. Micro., 63(9):3374-3377(1997)). Listeria monocytogenes bacteriophage can be combined with otherListeria monocytogenes specific bacteriophage to further refine theepidemiological typing results. The specificity of the Listeriamonocytogenes bacteriophage for certain strains of Listeriamonocytogenes demonstrates the utility of Listeria monocytogenesbacteriophage as an epidemiological typing tool.

The invention now will be exemplified in the following non-limitingexamples.

EXAMPLES Example 1 Listeria monocytogenes Bacteriophage Isolation

Listeria monocytogenes bacteriophage, specifically including LIST-1,LIST-2, LIST-3, LIST-4, LIST-36, and LIST-38, were isolated fromBaltimore Inner Harbor waters using lysis of Listeria monocytogenes toform plaques in bacterial lawns as a means of detecting the presence ofbacteriophage having lytic specificity for Listeria monocytogenes.Plaques are harvested, diluted and re-plated on bacterial lawns througha process of serial enrichment until a single bacteriophage species, ormonophage, results as determined by a stable restriction fragment lengthprofile of the bacteriophage DNA. The isolates obtained using thetechnique recited supra may be cultured using the techniques as setforth herein. Listeria monocytogenes bacteriophage was deposited withthe ATCC, receiving ATCC Deposit Accession Nos. PTA-5372, PTA-5373,PTA-5374, PTA-5375, PTA-5376 and PTA-5377.

PFU concentration of the Listeria monocytogenes bacteriophage may bedetermined using techniques known in the art, such as, for example, thelytic reaction described by Marquet-Van der Mee, N. & A. Audurier, Appl.Environ. Micro., 61(1):303-309 (1995), herein incorporated by reference.Briefly, host Listeria monocytogenes cells are inoculated into LB broth(Difco) and incubated at 30° C. until the onset of log phase growth(approximately 3 to 5 hours). Culture plater are then inoculated byflooding of the surface of the Modified Oxford agar (MOX, Difco) orLuria-Bertani broth agar (Difco) with 2 to 3 mls of the broth culture.After removal of the surplus inoculum, the plates are allowed to dry forat least 30 min. at 37° C. The phage preparations are then applied tothe seeded agar plates. The plates are incubated overnight at 30° C. Thedetermination of the lytic specificity of Listeria monocytogenesbacteriophage for a particular Listeria monocytogenes strain isdetermined by observing the plates for clear plaques on a lawn ofbacterial growth.

Example 2 Production of Listeria monocytogenes Bacteriophage Lysate inLiquid Culture

Listeria monocytogenes Bacteriophage Culturing

Single aliquots of Listeria monocytogenes, stored in 70% LB broth/30%glycerol medium, were revived from a −80° C. freezer. The Listeriamonocytogenes culture was allowed to thaw at room temperature for 15-30min., followed by brief vortexing. Ten ml of Listeria monocytogenes wereinoculated into 35 ml of LB-broth medium, and cultured at 30° C. at 150rpm over-night on a rotary shaker. The resulting OD₆₀₀ of the culturewas approximately 0.3-0.4.

Ten ml of Listeria monocytogenes were inoculated into 100 ml of LB brothmedium, and cultured at 30° C. at 150 rpm for approximately 2-2.5 hours,until the OD₆₀₀ reaches 0.1. To this culture were added a total ofapproximately 10⁹ PFU of Listeria monocytogenes bacteriophage. PFU ofthe Listeria monocytogenes bacteriophage was confirmed before-hand.

The mixture was then transferred to a 2 L flask containing 1.0 L of LBbroth. The mixture was cultured at 30° C. at 150 rpm for approximately5-7 hours, until the OD₆₀₀ reaches 0.04-0.01. At this point, Listeriamonocytogenes bacteriophage were harvested and purified.

Alternatively phage propagation can be carried out in 1 to 5 L flaskscontaining appropriate liquid microbiologic media, or in fermenterscontaining appropriate liquid microbiologic media. Batch fermentation iscarried out in sterilized fermentation equipment in volumes ranging from5 to 2,500 liters. A volume of an overnight culture in LB, TerrificBroth (TB) or similar rich bacteriological medium free of animalderivatives such as bovine albumin of the desired host strain ofListeria monocytogenes is incubated with a pre-determined optimal volumeof Listeria monocytogenes bacteriophage seed stock. Fermentation iscarried out at 30° C. to 37° C. for 5-7 h with periodic or continuousmonitoring of the OD₆₀₀ until optimal lysis and phage yield for eachhost-bacteriophage pair has occurred. Listeria monocytogenes strainswhich are lysed by the respective phage may be used for propagation (seeTable 2). Each of the 6 Listeria monocytogenes bacteriophage has lyticspecificity for Listeria monocytogenes ATCC number 35152 (serotype1/2a), ATTC number 19118 (serotype 4e) and ATCC number 15313 (serotype1/2a), each of which can be obtained from the ATCC using the ATCCDeposit Accession Number.

TABLE 2 Phage Listeria monocytogenes Strains Lysed by Phage List-1 Lm-4,Lm-26, Lm-54, Lm-91, Lm-107, Lm-171 List-2 Lm-30, Lm-88, Lm-89, Lm-102,Lm-122, Lm-174 List-3 Lm-38, Lm-51, Lm-54, Lm-83, Lm-101, Lm-142, Lm-107List-4 Lm-33, Lm-38, Lm-52, Lm-78, Lm-119, Lm-120, Lm-107 List-36 Lm-27,Lm-90, Lm-101, Lm-117, Lm-140, Lm-155 List-38 Lm-37, Lm-71, Lm-94,Lm-143, Lm-146, Lm-152 The host strains for each phage are underlined.

Bacterial cell suspensions containing phage are cleared of bacteria andbacterial fragments by either low speed centrifugation (usually employedfor batches <10 liters), or by tangential flow filtration (usuallyemployed for batches >10 liters). Low speed centrifugation is carriedout at 8,000×g for 30 min at 4° C. Supernatant fluids containingListeria monocytogenes bacteriophage are then filtered through an inert0.45 μm pore size filter, and processed as described below. Instead ofcentrifugation, larger volumes are:

(1) cleared of bacteria and bacterial debris by tangential flowfiltration through 0.22 μM Durapore (Millipore, Inc., Bedford, Mass.)PVDF (or essentially equivalent) filter.

(2) All filtrates are next treated with DNase and RNase, each atconcentrations of 0.75 mg/L for 30-60 min at room temperature.

(3) Following nuclease digestion, the bacteriophage are collected,washed, concentrated and exchanged into phosphate-buffered saline bytangential flow filtration using a 100 kDa spiral-wound regeneratedcellulose filter (CDUF006LH. Millipore, Inc.)—or essentially equivalentfilter. The tangential flow filtration process removes mediumcomponents, digested nucleic acids and the nucleases.

(4) The 100 klDa filtration is then followed by filtration through aninert 0.22 μM filter. Batches are handled aseptically following the 0.22μM filtration.

The concentration of Listeria monocytogenes bacteriophage is determinedby titration. The concentration of Listeria monocytogenes bacteriophageis adjusted to a specific concentration between 10⁹ to 10¹¹ PFU/ml bydilution with buffer or by concentration by tangential flow filtration.The lytic activity of the final product is then determined by titration.Titrations are highly accurate and reproducible when performed against asingle Listeria monocytogenes bacterial strain, but not when performedagainst a mixed culture of strains. The final titer of Listeriamonocytogenes bacteriophage is calculated.

Following titration, Listeria monocytogenes bacteriophage may befreeze-dried or spray-dried after addition of 10% skim milk or similarexcipient, or may be maintained and used in liquid form. An appropriatevolume of diluent may be added to achieve the specified final workingconcentration. The required volume can be validated for each lot ofListeria monocytogenes bacteriophage by reconstitution of test samplesand determination of the lytic titer of the bacteriophage determined asdescribed above.

Example 3 Alternative Production of Listeria monocytogenes BacteriophageLysate in Liquid Culture

Shake flask batches of each phage are produced in 2-L flasks rotated at100 to 200 rpm in a shaker-incubator (Model C-24; Now BrunswickScientific Co., Edison, N.J.). Listeria monocytogenes strains are grownin Luria-Bertani (LB) broth at 30° C. to an OD₆₀₀ of 0.1-0.3 absorbanceunits. Cultures are then infected at a multiplicity of infection (MOI;the ratio of phage to bacteria) previously determined to be optimal foreach phage. Growth is monitored spectrophotometrically until lysisoccurs and the phages are harvested by vacuum filtration (Stericup;Millipore, Billerica, Mass.). The material is then processed aspreviously described.

Large-scale batches of each phage are generated in a 10-L Bioflo 110fermenter (New Brunswick Scientific Co., Edison, N.J.) containing 10 Lof Terrific Broth (TB) supplemented with 4 ml/L glycerol and Antifoam204 (Sigma-Aldrich, St. Louis, Mo.) as needed up to a maximum of 200 μlAntifoam 204. The fermenter is inoculated with 100 ml of an activelygrowing seed culture in TR medium after the OD₆₀₀ of the culture isapproximately 1.0 (1×10⁹ CFU/ml). The fermenter is maintained at atemperature of 30° C., with an aeration rate of 3-7 L/min, a dissolvedoxygen level of ≧30% and a pH of 7.0±0.1. The pH is controlled byaddition of 1.2 N phosphoric acid (cat. no. PX0995-14; EMD Chemicals,Gibbstown, N.J.) or 1 N NaOH (cat. no. VW3225-6; EMD Chemicals,Gibbstown, N.J.), and foaming is controlled by addition of Antifoam 204as needed up to the previously stated maximum. Cultures are infected ata MOI of 0.01-0.5, based on a cell density of 1×10⁸ CFU/ml at an OD₆₀₀value of 0.1, when the OD₆₀₀ reaches the desired value. Likewise,infection is terminated when the OD₆₀₀ reaches the desired value. Thematerial is then processed as described previously.

Example 4 Application of Listeria monocytogenes Bacteriophage for thePreservation of Food Products

Listeria monocytogenes bacteriophage produced using the methods of thepresent invention may be dispersed in an appropriate aqueous solution orlyophilized or freeze-dried powder and applied to the surface of foodproducts. The data shown in FIGS. 1A-1L show that a mixture of equalparts of the six Listeria bacteriophage, List 1, List 2, List 3, List 4,List 36, and List 38, each at 1×10⁹ PFU/ml, achieves reductions ofgreater than 1 log when applied to the surfaces of ready to eatfoodstuffs at a density of 1 ml per 500 cm². The illustrated foodstuffswere previously contaminated with 2×10³ CFU per cm² of a mixture ofequal parts of Listeria monocytogenes strains ATCC 19115 (serogroup 4b),Lm 68 (serogroup 1/2b) and Lm 82 (serogroup 1/2a). The food samples wereincubated at room temperature for 20 min prior to application of theListeria bacteriophage, and were incubated at 4° C. for the indicatedtimes after the application of phage.

Alternatively, Listeria monocytogenes bacteriophage may be included witha cheese culture or other microbially active foodstuff prior to orduring processing. The Listeria monocytogenes bacteriophage are culturedfor a period of time on the surface of the food product or within thefood product.

Example 5 Isolation of Listeria monocytogenes Bacteriophage DNA

To isolate Listeria monocytogenes bacteriophage DNA, 0.75 ml of phage inphosphate-buffered saline solution (at a titer of 10⁸-10¹¹ PFU/ml) werecollected. To this phage were added 10 μl of proteinase K (20 mg/ml) and2 μl of RNase (10 mg/ml), followed by incubation at 37° C. for 30minutes, and a subsequent incubation at 56° C. for 30 minutes. Followingincubation, 75 μl of a mixture of 10% SDS (0.1 ml), 0.5 M EDTA (0.1 ml)and 0.8 ml of water were added and incubated at room temperature for 5min. To that mixture were added 0.75 μl of a phenol:chloroform:isoamylalcohol (25:24:1) solution, followed by centrifugation at 13,000 rpm forfive (5) min.

Next, the supernatant was carefully removed (approximately 600 μl), andtransferred to a clean eppendorf tube. Then, 0.6 ml of chloroform wereadded to the supernatant, mixed well, and centrifuged at 13,000 rpm forfive (5) min. The supernatant was then carefully extracted(approximately 500 μl).

Next, 0.1 volume of 3 M sodium acetate (40 ml) was added to thesolution, followed by 2.5 volumes of cold 95% ethanol (1 ml) toprecipitate the Listeria monocytogenes bacteriophage DNA. The solutionwas allowed to incubate at −20° C. for 1 hour, followed bycentrifugation at 13,000 rpm for thirty (30) min.

Following centrifugation, the pellet was washed with 1 ml of 70% coldethanol, and the supernatant was poured from the pellet. The pellet wasallowed to air dry, and was then resuspended in 36-360 μl of TE (10 mMtris-HCL, pH=8.5, 1 mM EDTA).

Example 6 Restriction Fragment Length Polymorphism (RFLP) Profile

DNA was isolated from Listeria monocytogenes bacteriophage using QiagenPlasmid Miniprep or Midiprep kits (Valencia, Calif.) according to themanufacturer's directions. Briefly, the instructions are as follows:

Harvest a desired quantity of Listeria monocytogenes bacteriophage bycentrifugation at 30,000×g for 2 to 3 h at 4° C. Resuspend the pelletedListeria monocytogenes bacteriophage in 250 μl buffer P1 (10 mMtris-HCl, pH=8, 100 μg/ml RNaseA) and transfer to a microcentrifugetube. Ensure that 100 μl/ml RNase A has been added to buffer P1. No cellclumps should be visible after resuspension of the pellet. Add 250 μl ofbuffer P2 (0.2 M NaOH, 2% SDS) and gently invert the tube 4-6 times tomix. Do not vortex, as this will result in shearing of genomic DNA. Ifnecessary, continue inverting the tube until the solution becomesviscous and slightly clear. Do not allow the lysis reaction to proceedfor more than 5 min.

Add 350 μl buffer N3 (4.2 M guanidine HCl, 0.9 M potassium acetate,pH=4.8) and invert the tube immediately but gently 416 times. To avoidlocalized precipitation, mix the solution gently but thoroughly,immediately after addition of buffer N3. The solution should becomecloudy. Centrifuge for 10 min at maximum speed in a tabletopmicrocentrifuge. A compact white pellet will form. Apply the supernatantto a plasmid DNA isolation spin column containing silica gel (i.e.,“QlAprep® column”) by decanting or pipetting. Centrifuge for 30-60 s.Discard the flow-through.

Wash the QIAprep column by adding 0.5 ml buffer PB (5 M guanidine HCl,30% isopropanol) and centrifuging for 30-60 s. Discard the flow-through.Wash QIAprep column by adding 0.75 ml buffer PE (80% ethanol/water) andcentrifuging for 30-60 seconds.

Discard the flow-through to allow for complete removal of the residualwash buffer, and centrifuge for an additional 1 min to remove residualwash buffer. Residual ethanol from buffer PE may inhibit subsequentenzymatic reactions. Place the QlAprep column in a clean 1.5 mlmicrocentrifuge tube. To elute DNA, add 50 μl buffer EB (10 mM Tris-Cl,pH=8.5) or water to the center of each QIAprep column, let stand for 1min, and centrifuge for 1 min. Substantially equivalent procedures arefollowed for isolation of bacteriophage DNA using the larger scalemidi-prep kit.

To perform the RFLP experiment with the isolated Listeria monocytogenesbacteriophage DNA, the following protocol is followed.

(1) Quantify the DNA by absorbance at 260 nm, and aliquot in amicrocentrifuge tube, 0.5-1 μg DNA per Listeria monocytogenesbacteriophage sample to be tested. Add, for example, 10 units Spel andmix, followed by an incubation at 37° C. for 2 hours.

(2) Add tracking dye (bromophenol blue+xylene cyanol) and separate on a1.0% agarose gel at 80 to 100 V for 50 min. Stain with ethidium bromide.Digestion with one or more additional enzymes (HindlIl, and/or EcoRV,and/or EcoRI) may be used if the RFLP patterns using Spel patterns areidentical to provide additional confirmation of identity.

Example 7 Lytic Specificity of Listeria monocytogenes bacteriophage

One hundred eighty L. monocyvogenes strains were screened forsusceptibility to a cocktail consisting of equal parts of LIST-1,LIST-2, LIST-3, LIST-4, LIST-36, and LIST-38 (identified as LMP-102) bythe drop on lawn spot test method. Strains were streaked onto LB agarand incubated at 37° C. overnight. Then, 10 μl of the mixture at 10⁹PFU/ml were dropped in triplicate on the bacterial streak. The plateswere incubated for 16 h at 37° C. and evaluated according to thefollowing criteria. If the zone of lysis was less than 1 mm, the resultwas considered negative. If the zone of lysis was between 1 and 3 mm indiameter with perceptible secondary growth of cells, the L.monocytogenes strain was considered moderately susceptible to lysis. Ifthe lysis zone equaled or exceeded 3 mm, the bacterial strain wasconsidered susceptible.

One hundred sixty strains (89%) of Listeria were susceptible to LMP-102,a cocktail of the six phage strains of interest, Table 3.

All references cited herein are herein incorporated by reference inentirety.

TABLE 3 Lytic specificity of Listeria bacteriophage for various Listeriaisolates. Suscep- Internal tibility Lab ID Serotype to LMP-102 Comments1 Lm-3 ½a + Environmental isolate 2 Lm-4 ½a + Environmental isolate 3Lm-5 ½a + Environmental isolate 4 Lm-6 ½a + Environmental isolate 5 Lm-7½a + Environmental isolate 6 Lm-8 ½a + Environmental isolate 7 Lm-9 ½a +Environmental isolate 8 Lm-10 4b + Environmental isolate 9 Lm-11 4a +Environmental isolate 10 Lm-12 ½a + Environmental isolate 11 Lm-13 ½a +Environmental isolate 12 Lm-14 ½a + Environmental isolate 13 Lm-15 ½a +Clinical isolate 14 Lm-17 4b + Clinical isolate 15 Lm-18 4b + Clinicalisolate 16 Lm-19 ½a + Clinical isolate 17 Lm-20 ½a − Clinical isolate 18Lm-21 4b + Clinical isolate 19 Lm-23 ½a + Clinical isolate 20 Lm-24 4b −Clinical isolate 21 Lm-25 ½a − Clinical isolate 22 Lm-26 ½a − Clinicalisolate 23 Lm-27 ½a + Environmental isolate 24 Lm-28 ½a − Environmentalisolate 25 Lm-29 4a + Environmental isolate 26 Lm-30 ND + Host strainfor List-2 phage; environmental isolate 27 Lm-32 4b + Environmentalisolate 28 Lm-33 ½a + Environmental isolate 29 Lm-34 ½a − Environmentalisolate 30 Lm-35 NT + Environmental isolate 31 Lm-37 4a + Clinicalisolate 32 Lm-38 ½a + Environmental isolate 33 Lm-39 ½a + Environmentalisolate 34 Lm-40 ½a + Environmental isolate 35 Lm-42 ½a − Environmentalisolate 36 Lm-43 ½a + Environmental isolate 37 Lm-45 ½a − Environmentalisolate 38 Lm-46 4b + Environmental isolate 39 Lm-49 NT + Environmentalisolate 40 Lm-50 ½a + Environmental isolate 41 Lm-51 4b + Clinicalisolate 42 Lm-52 4b − Clinical isolate 43 Lm-53 ½a + Clinical isolate 44Lm-54 ½a + Clinical isolate 45 Lm-55 ½a + Clinical isolate 46 Lm-56 ½a +Clinical isolate 47 Lm-57 3b − Clinical isolate 48 Lm-59 ½a + Clinicalisolate 49 Lm-61 ½b − Clinical isolate 50 Lm-62 ½a + Clinical isolate 51Lm-63 ½b + Clinical isolate 52 Lm-64 3b + Clinical isolate 53 Lm-65 ½b +Clinical isolate 54 Lm-66 ½b + Clinical isolate 55 Lm-67 ½b + Clinicalisolate 56 Lm-68 ½b + Clinical isolate 57 Lm-69 ½a + Clinical isolate 58Lm-70 ½a + Clinical isolate 59 Lm-71 ½a + Clinical isolate 60 Lm-72 4d +Clinical isolate 61 Lm-73 ½b + Clinical isolate 62 Lm-74 ½b + Clinicalisolate 63 Lm-75 ½b − Clinical isolate 64 Lm-76 ½b + Clinical isolate 65Lm-77 4b + Clinical isolate 66 Lm-78 4b + Clinical isolate 67 Lm-79 ½a +Clinical isolate 68 Lm-80 ½a ± Clinical isolate 69 Lm-81 4b + Clinicalisolate 70 Lm-82 ½a + CDC standard strain H2446; clinical isolate 71Lm-83 ½b + Clinical isolate 72 Lm-84 ½b + Clinical isolate 73 Lm-85 4b +Clinical isolate 74 Lm-86 ½b + Clinical isolate 75 Lm-87 ½a + Clinicalisolate 76 Lm-88 4a ± Clinical isolate 77 Lm-89 NT + Clinical isolate 78Lm-90 4b + Clinical isolate 79 Lm-91 ½a + Clinical isolate 80 Lm-92 ½a −Clinical isolate 81 Lm-93 ½a + Clinical isolate 82 Lm-94 4b − Clinicalisolate 83 Lm-95 ½a + Clinical isolate 84 Lm-96 4b + Environmentalisolate 85 Lm-97 ½a + Clinical isolate 86 Lm-98 ½a + Clinical isolate 87Lm-99 4b + Clinical isolate 88 Lm-100 ½c + Clinical isolate 89 Lm-101½c + Clinical isolate 90 Lm-102 ND + Clinical isolate 91 Lm-103 4b +Clinical isolate 92 Lm-104 4b + Clinical isolate 93 Lm-105 ½a + Clinicalisolate 94 Lm-106 ½a + Clinical isolate 95 Lm-107 ½a + Host strain forList-1, List-3 and List-4 phages; clinical isolate 96 Lm-108 4b +Clinical isolate 97 Lm-109 ½c + Clinical isolate 98 Lm-110 ½a + Clinicalisolate 99 Lm-111 ½a + Clinical isolate 100 Lm-112 ½c + Clinical isolate101 Lm-113 ½a + Clinical isolate 102 Lm-114 4b + Environmental isolate103 Lm-115 4b + Clinical isolate 104 Lm-116 4b + Clinical isolate 105Lm-117 4b + Host strain for List-36 phage; clinical isolate 106 Lm-1184b + Clinical isolate 107 Lm-119 ½a + Environmental isolate 108 Lm-1204b + Environmental isolate 109 Lm-121 ½a + Environmental isolate 110Lm-122 ½a + Environmental isolate 111 Lm-123 ½a + Environmental isolate112 Lm-124 NT + Environmental isolate 113 Lm-125 4b + Environmentalisolate 114 Lm-126 ½b + Environmental isolate 115 Lm-127 ½a +Environmental isolate 116 Lm-128 ½b + Environmental isolate 117 Lm-129½a + Environmental isolate 118 Lm-130 ½a + Environmental isolate 119Lm-131 ½a + Clinical isolate 120 Lm-132 ½b ± Clinical isolate 121 Lm-133NT + Clinical isolate 122 Lm-134 NT + Clinical isolate 123 Lm-135 ½a +Clinical isolate 124 Lm-136 ½a + Clinical isolate 125 Lm-137 ½a +Clinical isolate 126 Lm-138 NT + Clinical isolate 127 Lm-139 ½a +Clinical isolate 128 Lm-140 ½a + Clinical isolate 129 Lm-141 ½a +Clinical isolate 130 Lm-142 ½a + Environmental isolate 131 Lm-143 ½b +Environmental isolate 132 Lm-144 4b + Environmental isolate 133 Lm-145½a + Environmental isolate 134 Lm-146 ½a + Host strain for List-38phage; environmental isolate 135 Lm-147 ½a + Environmental isolate 136Lm-148 ½a − Environmental isolate 137 Lm-149 ½a − Environmental isolate138 Lm-150 ½a + Environmental isolate 139 Lm-151 ½a + Environmentalisolate 140 Lm-152 ½a + Environmental isolate 141 Lm-153 ½a +Environmental isolate 142 Lm-154 ½a + Environmental isolate 143 Lm-155½a + Environmental isolate 144 Lm-156 ½a + Environmental isolate 145Lm-157 ½a + Environmental isolate 146 Lm-158 ½a + Environmental isolate147 Lm-159 4a + Environmental isolate 148 Lm-160 ½a + Environmentalisolate 149 Lm-161 ½a + Environmental isolate 150 Lm-162 ½a +Environmental isolate 151 Lm-163 ½a + Environmental isolate 152 Lm-164½a + Environmental isolate 153 Lm-165 ½a + Clinical isolate 154 Lm-166½a + Clinical isolate 155 Lm-167 4b + Clinical isolate 156 Lm-168 4b +Clinical isolate 157 Lm-169 ½a + Clinical isolate 158 Lm-170 ½a +Clinical isolate 159 Lm-171 ½b + Clinical isolate 160 Lm-172 ½a +Clinical isolate 161 Lm-174 ½a + Environmental isolate 162 Lm-175 ½a +Environmental isolate 163 Lm-176 NT − Environmental isolate 164 Lm-1774b + Environmental isolate 165 Lm-178 4b + Clinical isolate 166 Lm-183NT + Environmental isolate 167 Lm-184 ½a + Environmental isolate 168Lm-185 NT + Environmental isolate 169 Lm-191 ½a + Environmental isolate170 Lm-192 ½a − Environmental isolate 171 Lm-193 3a + Unknown 172 Lm-194NT + Unknown 173 Lm-195 ½a + Unknown 174 Lm-196 ½a + Unknown 175 Lm-198½a + Unknown 176 Lm-200 ½b − Unknown 177 Lm-201 ½b + Unknown 178 Lm-300½c + Environmental isolate 179 Lm-301 ½a + Environmental isolate 180Lm-302 4b + Environmental isolate Clinical isolate designates an isolatefrom a patient with listeriosis, or from an asymptomatic carrier NA =not available, PFGE typing performed, but PFGE type not yet assigned NT= Not Typeable + Lysis zone ≧3 mm, clear of secondary colonies ± Lysiszone 1-3 mm, with some visually detectable secondary growth of cells −Lysis zone ≦3 mmThe table may be summarized as follows:

-   -   17 (9.4%) strains are resistant to LMP-102™    -   3 (1.7%) strains are moderately susceptible to LMP-102™    -   160 (88.9%) strains are susceptible to LMP-102™

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

1. A substantially pure bacteriophage composition comprising one or moreListeria monocytogenes bacteriophage strains selected from the groupconsisting of phage having ATCC accession nos. PTA-5372, PTA-5373,PTA-5374, PTA-5375, PTA-5376 and PTA-5377.
 2. The composition of claim 1containing at least two of said strains.
 3. The composition of claim 1containing at least three of said strains.
 4. The composition of claim 1containing at least four of said strains.
 5. The composition of claim 1containing at least five of said strains.
 6. The composition of claim 1containing six of said strains.